The fibroblast growth factor (FGF) family of proteins comprises at least 22 polypeptides, referred to as FGF1-FGF22, with diverse biological activities. For reviews, see, McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 1998, 59:135-176; Nishimura et al., Biochim Biophys. Acta. 2000, 1492:203-206; Yamashita et al., Biochem. Biophys. Res. Commun. 2000, 277:494-498. For example, FGF polypeptides modulate the proliferation and differentiation of a variety of cells of mesenchymal and neuro-ectodermal origin (Basilico & Moscatelli, Adv. Cancer Res. 1992, 59:115-165). FGF polypeptides also play critical roles during embryonic processes such as mesoderm induction, post-implantation blastocyst development, and limb and lung development (Goldfarb, Cytokine Growth Factor Rev. 1996, 7:311-325; Xu et al., Cell Tissue Res. 1999, 296:33-43). Increased FGF signaling leads to a variety of human skeletal disorders, including dwarfism and craniosynostosis syndromes (McIntosh et al., Cell Struct. Funct. 2000, 25:85-96; Naski & Omitz, Front. Biosci. 1998, 3:D781-D794; Wilkie, Hum. Mol. Genet. 1997, 6:1647-1656). In adult organisms FGFs are thought to be involved in physiological angiogenesis and wound healing as well as in pathological angiogenesis such as in tumor neovascularization and diabetic retinopathy (Basilico & Moscatelli, Adv. Cancer Res. 1992, 59:115-165).
The diverse effects of FGFs are mediated by at least four receptor tyrosine kinases, which are referred to collectively as the FGF receptor (FGFR) polypeptides and are known individually as FGFR1-FGFR4. The FGFR polypeptides comprise an extracellular domain, a single transmembrane helix and a cytoplasmic portion. The extracellular domain binds to the FGF polypeptide ligand, and may be subdivided into at least three distinct three immunoglobulin (Ig)-like domains, known as D1-D3, with each domain being connected by a “linker” polypeptide sequence. Ligand binding and specificity resides in the D2 and D3 domains and the short D2-D3 linker (Plotnikov et al., Cell 1999, 98:641-650; Plotnikov et al., Cell 2000, 101:413-424; Stauber et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97:49-54).
FGFR dimerization is prerequisite for FGF signaling and requires heparin or heparan sulfate proteoglycans (HSPG) (Ornitz, Bioessays 2000, 22:108-112; Schlessinger, Cell 2000, 103:211-225). The recent crystal structure of a ternary FGF2-FGFR1-heparin complex has provided a mechanistic view of the process by which heparin aids FGF polypeptides to induce FGFR dimerization (Schlessinger et al., Mol. Cell 2000, 6:743-750). According to this “two end” model, heparin interacts via its non-reducing end with the heparin binding sites of FGF and FGFR and promotes the formation of a ternary 1:1:1 FGF:FGFR:heparin complex. A second ternary 1:1:1 FGF:FGFR:heparin complex is then recruited to the first ternary complex via interactions of FGFR, FGF and heparin in one ternary complex with the FGFR in the adjoining ternary complex.
A fundamentally different model for FGFR dimerization has emerged from the recent crystal structure of a dimeric FGF1-FGFR2-heparin ternary complex (Pellegrini et al., Nature 2000, 407:1029-1034). In this structure, a single heparin molecule links two FGF ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF molecules but only one receptor chain. There is essentially no protein-protein interface between the two 1:1 FGF-FGFR complexes in the dimer.
With the exception of FGF1, which is the universal ligand for all FGFRs, most FGF polypeptides exhibit specific, albeit promiscuous, patterns of receptor binding affinity (Ornitz et al., J. Biol. Chem. 1996, 271:15292-15297). Comparison of the crystal structures of FGF1-FGFR1, FGF2-FGFR1 and FGF2-FGFR2 complexes defined a general binding interface for FGF-FGFR complexes involving contacts made by FGF to D2 and to the D2-D3 linker (Plotnikov et al., Cell 2000, 101:413-424). It has also been shown that specificity is achieved through interactions of the FGF N-terminal (i.e., the amino acid sequence immediately preceding the FGF polypeptide's β-trefoil core domain) and central regions with FGFR D3. These structures have also provided a molecular basis for how alternative splicing in FGFR modulates specificity. In both FGF2-FGFR1 and FGF2-FGFR2 structures, FGF2 makes specific contacts with the βC′-βE loop in D3, which is subject to alternative splicing. Consequently, FGF2 discriminates between the IIIc and IIIb variants of FGFRs. In contrast, FGF1 does not interact with the βC′-βE loop and therefore can bind all FGFRs irrespective of alternative splicing in D3 (Plotnikov et al., Cell 2000, 101:413-424).
FGF4 shares about 30% sequence identity with the prototypical members of the FGF family, FGF1 and FGF2 (Delli Bovi et al., Cell 1987, 50:729-737). FGF4, unlike FGF1 and FGF2, has a classical signal peptidp and thus is efficiently secreted from cells (Bellosta et al., J. Cell Biol. 1993, 121:705-713). Most receptor binding studies indicate that FGF4 binds and activates the IIIc splice forms of FGFR1-3 to comparable levels, but it shows little activity towards the IIIb splice forms of FGFR1-3 as well as towards FGFR4 (Ornitz et al., J. Biol. Chem. 1996, 271:15292-15297; Vainikka et al., EMBO J. 1993, 11:4273-4280). As for FGF1 and FGF2, heparin greatly augments the biological activity of FGF4 on cells lacking endogenous cell surface HSPG (Mansukhani et al., Proc. Natl. Acad. Sci. U.S.A. 1992, 89:3305-3309). However, employing selectively O-desulfated heparins, Guimond et al. (J. Biol. Chem. 1993, 268:23906-23914) have shown that both 2-O-and 6-O-desulfated heparin were able to support the mitogenic activity of FGF4, while neither of these heparins could support the biological activity of FGF1 and FGF2. It has therefore been suggested the sulfation motifs in heparin required for FGF4 activity may differ from those required for FGF1 and FGF2 actions (Guimond et al., supra; Ishihara, Glycobiology 1994, 4:817-824).
In summary, the exact interactions that stabilize complexes of the FGF4 polypeptide with its receptor and/or heparin remain poorly understood. Yet, given the range of biological disorders associated with FGF signaling, there is an urgent need to identify and characterize these interactions. There exist, moreover, a need to identify compounds that modulate binding of FGF4 to either an FGF receptor or heparin, including mutant or variant forms of the FGF4 polypeptide that have altered binding affinities, as well as other compounds that may be agonists or antagonists of FGF4 binding and/or activity.